Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates
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Evaluation Summary:
The ribonucleoprotein YBX1 is required for sorting of miR-233 to exosomes. Here, the authors demonstrate that YBX1 undergoes liquid liquid phase separation (LLPS) both in vitro and in vivo and YBX1 droplets specifically partition miR-233 and mediate its packaging into exosomes. The authors also demonstrate a possible connection between YBX1 condensates and P-bodies during cargo sorting into EVs. Overall, this is an elegant study and with a few additional experiments to clarify the involvement of P bodies, the story will be of broad impact to those interested in LLPS and RNA packaging into exosomes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Exosomes may mediate cell-to-cell communication by transporting various proteins and nucleic acids to neighboring cells. Some protein and RNA cargoes are significantly enriched in exosomes. How cells efficiently and selectively sort them into exosomes remains incompletely explored. Previously, we reported that YBX1 is required in sorting of miR-223 into exosomes. Here, we show that YBX1 undergoes liquid-liquid phase separation (LLPS) in vitro and in cells. YBX1 condensates selectively recruit miR-223 in vitro and into exosomes secreted by cultured cells. Point mutations that inhibit YBX1 phase separation impair the incorporation of YBX1 protein into biomolecular condensates formed in cells, and perturb miR-233 sorting into exosomes. We propose that phase separation-mediated local enrichment of cytosolic RNA-binding proteins and their cognate RNAs enables their targeting and packaging by vesicles that bud into multivesicular bodies. This provides a possible mechanism for efficient and selective engulfment of cytosolic proteins and RNAs into intraluminal vesicles which are then secreted as exosomes from cells.
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Evaluation Summary:
The ribonucleoprotein YBX1 is required for sorting of miR-233 to exosomes. Here, the authors demonstrate that YBX1 undergoes liquid liquid phase separation (LLPS) both in vitro and in vivo and YBX1 droplets specifically partition miR-233 and mediate its packaging into exosomes. The authors also demonstrate a possible connection between YBX1 condensates and P-bodies during cargo sorting into EVs. Overall, this is an elegant study and with a few additional experiments to clarify the involvement of P bodies, the story will be of broad impact to those interested in LLPS and RNA packaging into exosomes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name …
Evaluation Summary:
The ribonucleoprotein YBX1 is required for sorting of miR-233 to exosomes. Here, the authors demonstrate that YBX1 undergoes liquid liquid phase separation (LLPS) both in vitro and in vivo and YBX1 droplets specifically partition miR-233 and mediate its packaging into exosomes. The authors also demonstrate a possible connection between YBX1 condensates and P-bodies during cargo sorting into EVs. Overall, this is an elegant study and with a few additional experiments to clarify the involvement of P bodies, the story will be of broad impact to those interested in LLPS and RNA packaging into exosomes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Extracellular vesicles (EVs) are critical for intercellular communication by delivering signal molecules, miRNAs, and also nutrients to recipient cells. The mechanisms on selective sorting of cargos into EVs are still largely unknown. The authors previously revealed that the ribonucleoprotein (RBP) YBX1 is required for sorting of miR-233 to exosomes. In this manuscript, the authors further investigated the detailed mechanism. By conducting extensive imaging, fractionation, and biochemical-based experiments, they convincingly demonstrate that YBX1 undergoes liquid liquid phase separation both in vitro and in vivo through its C-terminal IDR. The YBX1 droplets specifically partition miR-233 and mediate its packing into exosomes. Interestingly, the authors demonstrate a close connection between YBX1 condensates …
Reviewer #1 (Public Review):
Extracellular vesicles (EVs) are critical for intercellular communication by delivering signal molecules, miRNAs, and also nutrients to recipient cells. The mechanisms on selective sorting of cargos into EVs are still largely unknown. The authors previously revealed that the ribonucleoprotein (RBP) YBX1 is required for sorting of miR-233 to exosomes. In this manuscript, the authors further investigated the detailed mechanism. By conducting extensive imaging, fractionation, and biochemical-based experiments, they convincingly demonstrate that YBX1 undergoes liquid liquid phase separation both in vitro and in vivo through its C-terminal IDR. The YBX1 droplets specifically partition miR-233 and mediate its packing into exosomes. Interestingly, the authors demonstrate a close connection between YBX1 condensates and P-bodies during cargo sorting into EVs. Overall, this is an elegant, well-conducted and-controlled study.
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Reviewer #2 (Public Review):
Using a variety of cell biological and biochemical techniques, Liu et al. demonstrate that the RNA-binding protein YBX1 forms biomolecular condensates in cells and in vitro. Condensation is required for loading of YBX1 and miR-233 into exosomes, while other miRNAs are not recruited. This is a great example how liquid-liquid phase separation of an RNA-binding protein can selectively recruit a specific RNA, both in vitro and in cells, and how condensation is essential for the targeting and 'function' of this RNP complex. Furthermore, the authors demonstrate that certain P-body factors are co-recruited into MVBs, revealing a connection between these membraneless and membrane-bound organelles.
Overall, the conclusions of the manuscript are well supported by the experiments, and the experiments very cleanly …
Reviewer #2 (Public Review):
Using a variety of cell biological and biochemical techniques, Liu et al. demonstrate that the RNA-binding protein YBX1 forms biomolecular condensates in cells and in vitro. Condensation is required for loading of YBX1 and miR-233 into exosomes, while other miRNAs are not recruited. This is a great example how liquid-liquid phase separation of an RNA-binding protein can selectively recruit a specific RNA, both in vitro and in cells, and how condensation is essential for the targeting and 'function' of this RNP complex. Furthermore, the authors demonstrate that certain P-body factors are co-recruited into MVBs, revealing a connection between these membraneless and membrane-bound organelles.
Overall, the conclusions of the manuscript are well supported by the experiments, and the experiments very cleanly performed and well controlled. This work is a great starting point in dissecting which proteins and RNAs are loaded into exosomes, how condensation of one protein contributes to the recruitment of itself but also other RNA and protein factors, and puts forward several follow-up research questions (e.g. RNA selectivity, the connection to P-bodies).
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Reviewer #3 (Public Review):
How cells signal each other is one of the key questions in biology and for the understanding of tissue's disease states like cancer. Whereas much work focused on classic receptor kinase pathways, comparatively little is known about other means of signaling.
Interestingly, cells can secrete - among many other molecules - RNA and although it has been under debate whether secreted RNA is indeed found inside secreted membrane bound vesicles it has been suggested that secretion of RNA could be a powerful way of signaling neighboring cells their own disease state.
Previous work from the Schekman lab demonstrated that a small class of RNA (miRNA) becomes enriched in biochemical fractions of exosomal markers. Although miRNA have been found in exosomal preparations before, Shurtleff et al. 2016 developed an advanced …
Reviewer #3 (Public Review):
How cells signal each other is one of the key questions in biology and for the understanding of tissue's disease states like cancer. Whereas much work focused on classic receptor kinase pathways, comparatively little is known about other means of signaling.
Interestingly, cells can secrete - among many other molecules - RNA and although it has been under debate whether secreted RNA is indeed found inside secreted membrane bound vesicles it has been suggested that secretion of RNA could be a powerful way of signaling neighboring cells their own disease state.
Previous work from the Schekman lab demonstrated that a small class of RNA (miRNA) becomes enriched in biochemical fractions of exosomal markers. Although miRNA have been found in exosomal preparations before, Shurtleff et al. 2016 developed an advanced purification protocol which excluded the presence of non-membrane bound "contaminants", among them RNA binding protein complexes. They as well demonstrated the presence of miRNA inside vesicles and identified in them the protein YBX1 - which was found by mass spec to be enriched in exosomal preps - to be responsible for exosomal enrichment of certain miRNA. YBX-1 was identified by RNA affinity chromatography and its overexpression is linked to cancer. Previously, it was thought that Argonautes could be involved in miRNA targeting, but that could be ruled out now.
In this manuscript by Liu et al., the authors now study how YBX-1 can specifically sort miRNA into exosomes and come to the conclusion that condensation of YBX-1 by phase separation could be the mechanism to enrich not only YBX-1 but as well the specific RNA type miR-223 in exosomes.
They find that YBX-1 forms liquid-like condensates in cells which are sensitive to treatment with 1,6 hexanediol, find that these condensates can fuse and turn over quickly to 70% of fluorescence after FRAP and show that the C-terminal IDR is responsible for condensate formation. When analyzing the IDR further, they find that YBX-1 IDR mutants, in which either all tyrosines or all arginines and lysines were replaced, showed lower enrichment of miR-223 compared to wt YBX-1 (which is in the range of 100-1000 fold depending on the cell type; see Fig 5A).
The respective YBX-1 IDR mutant Y to S mutant formed smaller condensates in vitro but still recruited miR-223, whereas the RK to G mutant did not form condensates, indicating that RNA binding and condensate formation are mediated by different domains. miR-223 recruitment is mediated by the cold shock domain (CSD), a domain described in LIN28 to recruit LIN7 miRNA. A conserved phenylalanine residue in the CSD (F85 in YBX-1) has previously been shown to be required for RNA binding.
Overall, this manuscript is very well written, the experiments are well described and the quality of the data is very good. Although several of the experiments seem at first sight not to be novel, the quality of this manuscript is that these experiments are combined to a new idea, which is how cells could make use of condensate biology to extract certain molecules from P bodies and secrete them to neighboring cells.
The YBX-1 mutant analysis is pretty convincing, demonstrating that the aromatic or basic charges are required to sort YBX-1 into exosomes. Previous work has shown that aromatic and basic residues play a role in condensate formation of RNA binding proteins with PLD's (Wang et al., 2018). Importantly, the F85A mutant is not affected in condensate formation in vitro, showing that miRNA is not involved in condensate formation but rather becomes enriched in exosomes by association with the CSD domain.
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